- OBJECTIVE: We sought to confirm the finding of pepsin/pepsinogen in the middle ear fluid of children with otitis media in a larger sample size using a sensitive and specific pepsin assay. STUDY DESIGN AND SETTING: We evaluated 152 children (225 ear samples) in a prospective study at a tertiary care children's hospital. The presence of pepsin in middle ear aspirates was determined using enzymatic assay. RESULTS: Of the patients, 14.4 percent (22 of 152) had detectable pepsin activity in one or both of the ear samples with no pepsin activity detected in control serum. Average pepsin concentration in the samples was 96.6 +/- 170.8 ng/ml, ranging from 13 to 687 ng/ml. Pepsin concentration in the middle ear of children younger than 1.0 year was significantly higher than in older age groups. CONCLUSION AND SIGNIFICANCE: Results indicate that pepsin/pepsinogen is present in the middle ears of children with otitis media, although not at the high rate previously reported. Gastric reflux may be one causative factor in the pathogenesis of otitis media.